ProjectSummary The goal of this project is to understand how brachyury expression is maintained in chordoma and to identify approaches to attack brachyury function as a therapeutic strategy. Chordoma is a rare primary tumor that develops in the skull base and spine and currently has no targeted therapies. Strong evidence supports the hypothesisthatchordomaisdrivenbyexpressionoftheTgene,whichencodestheproteinbrachyury.Brachyury isatranscriptionfactor(TF)thatisresponsibleforcontrollingthegenesrequiredformesodermandnotochord formation.Brachyuryexpressionisnormallyturnedoffearlyindevelopment,however,chordomacellsfailtoturn offtheTgene,resultinginsustainedbrachyuryexpression.Withinourpreviouswork,wehavemappedacluster super-enhancers adjacent to the T gene that we believe are necessary to drive this aberrant brachyury expression. Genetic knockout of brachyury arrests chordoma cells, which suggests that inhibiting brachyury functionpresentsapromisingtherapeuticstrategy.However,historicallytranscriptionfactorshavebeendifficult to directly target because, unlike other proteins that contain active sites, transcription factors lack obvious pockets for small molecule ligand binding. The overarching goal of this project is to gain insight into how brachyuryexpressionispropagatedandtoidentifyothereffectiveapproachesfortargetingbrachyuryfunction inconjunctionwithitsdirectinhibition.Wehypothesizethatwecaninhibitbrachyuryexpressionupstream ofitstranscriptionalactivationasatherapeuticstrategy.Totestthishypothesis,wewillengineeramodel systemwherecompletebrachyurydegradationcanbeachievedtomodeltheconsequencesofitsperturbation. Examining the consequences of brachyury degradation in comparison to inhibiting its activation upstream will elucidate the efficacy between direct and indirect brachyury inhibition. Finally, functionally dissecting the brachyurysuper-enhancerstoidentifythecriticalregionsfortrans-factorbindingmayelucidatenovelchordoma therapeutictargets.Together,theseproposedstudieswilllendinsightintotheroleofbrachyuryinchordomaand willhelptoachievethecommongoalofdevelopingnewapproachestotherapeuticallytargetbrachyury.